The Study On The Effect Of Streptococcus Mitis On The Pathogenicity Of Pseudomonas Aeruginosa And Its Mechanisms

PART ⅠThe effect of Streptococcus mitis on the pathogenicity of Pseudomonas aeruginosa biofilmObjectives: The ventilator-associated pneumonia(VAP) remains a serious problem in newborns due to biofilm formation on the endotracheal tube(ETT). We previously found that the Pseudomonas aeruginosa(P.aeruginosa) and Streptococcus mitis(S. mitis), the normal colonized bacteria in oropharynx, were the most common bacteria in the biofilm(BF)on newborns’ ETT, and the detection rate of S. mitis was higher in the VAP group. Since the microorganisms in the biofilm might promote or suppress each other, the present study was to investigate whether S. mitis could influence the pathogenicity of P. aeruginosa.Methods: The PAO1 and(or) S. mitis were cultured and the biofilm conditioned medium(BCM) was collected. The adhesion was measured by crystal violet assay, plate counting method and light microscopy. The BF formation and live bacteria were measured by crystal violet assay,plate counting method, cell counting kit-8(CCK-8) and confocal laserscanning microscopy(CLSM). Human airway epithelial(BEAS-2B) cells were cultured and stimulated by BCM, and the cell viability and the expression of TLR2/4 and IL-8 were measured.Results: We found that S. mitis alone barely adhered to the abiotic surface and couldn’t form biofilm, but it enhanced the adhesion and BF formation of PAO1(P<0.05). The number of total live bacteria and live P.aeruginosa in the mixed BF was more than that of PAO1 BF(P<0.05);nevertheless, the host immune response induced by the mixed BCM was alleviated compared to that of PAO1 BCM(P<0.05).Conclusions: We found that S. mitis, as a the normal colonized bacteria in oropharynx, enhanced the adhesion and biofilm formation of P.aeruginosa; nevertheless, it alleviated the immune response induced by P.aeruginosa. These findings remind us that we should not ignore the role of tranditional-viewed non-pathogenic bacteria in the biofilm, and provide new insights into exploring the bacteria interaction mechanisms in neonatal VAP or other biofilm related infection.PART ⅡThe effect of Streptococcus mitis on the the pathogenicity of planktonic Pseudomonas aeruginosaObjectives: We demonstrated in the PART Ⅰ that S. mitis enhanced the adhesion and biofilm formation of PAO1 and it alleviated the immune response induced by PAO1. The matured BF could release planktonic bacteria to attack the host and induce the immune response. Moreover, it is reported that some normal colonized bacteria in oropharynx(such as Porphyromonas gingivalis, actinomycetes) could enhance the adhesion ability to the targeted cells or tissue, thus influencing the pathogenicity of P. aeruginosa. Therefore, we wonder whether the S. mitis could influence the pathogenicity of planktonic P. aeruginosa ? In this part, we would prove this problem.Methods: We divided the bacteria into three groups: PAO1 group, S.mitis group and PAO1 + S. mitis group. Then the bacteria was added to the BEAS-2B cells(MOI=100). The invasion and adhesion ability were observed by the invasion and adhesion assay. The cell viability was detected by CCK-8 method, and the expression of IL-8 was measured by ELISA.Results: Compared the PAO1 group, S. mitis could the enhance the invasion ability of PAO1 to the BEAS-2B cells(P<0.05). However, it showed no significant differences in the adhesion ability between the PAO1 + S. mitis group and PAO1 group(P>0.05). The cell viability and the expression of IL-8 showed no significant differences(P>0.05).Conclusions: The S. mitis could the enhance the invasion ability of PAO1 to the BEAS-2B cells, but it showed no effect to the adhesion ability,indicating that the mechanism between the invasion and adhesion is different. Although the invasion ability of planktonic PAO1 was enhanced due to S. mitis, the cell viability and IL-8 expression were not influenced,which might be beneficial for the P. aeruginosa BF formation inside the cells and protect the P. aeruginosa from the host immune attack. The influence of S. mitis on the PAO1 pathogenicity probably mainly rely on its BF state.PART ⅢStreptococcus mitis enhance the biofilm formation via upregulating the QS system of Pseudomonas aeruginosaObjectives: The communication in the multi-species BF is important in the BF formation, among which quorum sensing(QS) is a key link. We have demonstrated that S. mitis enhanced the adhesion and biofilm formation of PAO1. However, the mechanisms remained unclear. Quorum sensing(QS) system plays a key role in bacterial adhesion and biofilm formation. Therefore, is the enhancement of S. mitis on P. aeruginosa related to the QS system of P. aeruginosa ?Methods: The BF groups were as follows: PAO1 group、P.a Δlas R group、P.a ΔLas RΔrhl R group、S. mitis group、PAO1+S. mitis group、P.a Δlas R+S. mitis group 、 and P.a ΔLas RΔrhl R+S. mitis group. The adhesion was measured by crystal violet assay, plate counting method and light microscopy. The BF formation and live bacteria was measured by crystal violet assay, plate counting method, and confocal laser scanning microscopy(CLSM). The expressions of QS genes(las I、las R、rhl I、rhl R)were detected by q PCRResults: The adhesion of PAO1+S.mitis、P.a Δlas R+S. mitis、and P.aΔLas RΔrhl R+S. mitis were enhanced compared with the PAO1、P.a Δlas R and P.a Δlas RΔrhl R group(P<0.05). Moreover, the plate counting method proved that the live P. aeruginosa in the PAO1+S.mitis 、 P.a Δlas R+S.mitis、and P.a ΔLas RΔrhl R+S. mitis group were more than those of thePAO1, P.a Δlas R, and P.a Δlas RΔrhl R group seperately(P<0.05). The biomass in the PAO1+S. mitis group was more than that of the PAO1group(P<0.05); however, there were no significant differences between the P.a Δlas R+S. mitis, P.a ΔLas RΔrhl R+S. mitis group and the P.a Δlas R,P.a Δlas RΔrhl R group(P>0.05). The S.mitis could upregulate the expression of QS genes(las I、las R、rhl I、rhl R) when co-cultured with PAO1 strain, compared with that of the PAO1 group(P<0.05).Nevertheless, the expression of QS genes were not upregulated in the P.aΔlas R+S. mitis, P.a ΔLas RΔrhl R+S. mitis group, compared with those of the P.a Δlas R、P.a Δlas RΔrhl R group(P>0.05).Conclusions: The increased adhesion of P. aeruginosa by S.mitis was independent of the QS system, implying some other mechanisms existed.The S. mitis enhance the BF formation of P. aeruginosa via upregulating the QS genes of P. aeruginosa. Still, we didn’t figure out that it is the S.mitis itself or some other substances it secreted that promote the BF formation. Future studies will be required to clarify the underlying mechanisms.